US5990286A - Antibodies with reduced net positive charge - Google Patents
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- US5990286A US5990286A US08/781,449 US78144997A US5990286A US 5990286 A US5990286 A US 5990286A US 78144997 A US78144997 A US 78144997A US 5990286 A US5990286 A US 5990286A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3046—Stomach, Intestines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3061—Blood cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/54—F(ab')2
Definitions
- the present invention relates generally to the field of modified antibodies. More specifically, the invention relates to chemically modified antibodies having increased binding specificity, improved pharmacokinetics and localization capabilities. These modified antibodies are particularly useful in the diagnosis and therapy of cancer and other mammalian disease.
- MAb's monoclonal antibodies
- MAb's specific to tumor cell antigens have been produced. It has also been shown that MAb's may be efficiently coupled to adjuncts such as radionuclides. Such radio-labelled MAb's are useful in providing clinical data, such as tumor imaging from immunoscintography, also known as g-camera imaging or radioimmunoimaging. In immunoscintography, the MAb's are allowed to bind to the specific tissue or tumor types having the antigen recognized by the MAb's. The radionuclides are then visualized through the use of appropriate technology, such as through the use of a germanium camera. It is the unique specificity of MAb's which enables their application in immunoscintography of tumors and other types of tissues.
- the cytotoxic effect of MAb's can be markedly increased by coupling to radionuclides, drugs or toxins.
- the unique specificity of MAb's has raised hopes of the development of immunotherapy.
- biologically active agents are delivered using MAb's to particular undesirable cell types, such as tumor cells, thereby affecting the undesirable cell types without affecting other cells of the subject.
- immunotherapies require extremely high specificity antibodies in order to avoid affecting healthy tissue.
- a method of increasing the specificity of MAb's would be highly beneficial in achieving the goal of a safe, effective immunotherapy.
- MAb's remain in the circulation for several days following introduction into a subject. This is undesirable for at least two reasons. One reason is that circulating MAb's produce high background levels in immunoscintography. A second reason is that circulating MAb's coupled to radionuclides or other potentially cytotoxic agents may produce undesirable side effects in the subject after prolonged exposure. Thus, there is a need for a method of decreasing the clearance time of MAb's. Of course, too great a decrease would result in MAb's being eliminated before any effective use of the MAb's could be made. Thus, there is a particular need for a method of decreasing the clearance time of MAb's without substantially affecting uptake of MAb's by tumor or other target tissue.
- an "intact" antibody molecule will refer to an unmodified antibody molecule comprised of two heavy chains and two light chains.
- the intact, whole antibody molecule is seen on the reactant side of the chemical equation of FIG. 1.
- the intact molecule is divided into the F c and the F ab domains.
- F(ab') 2 the bivalent form of the F ab fragmnent, may be produced through the digestion of the F c domain with a protease.
- the two heavy chains (designated as "H” in FIG. 1) are held together by one or more disulfide bridges. In intact molecules these disulfide bridges are normally protected from reducing agents. It has been found however, that removal of the F c domain allows facile reduction of the disulfide bridges. Thus, F(ab'), the monovalent form, may be produced from F(ab') 2 through the action of a mild reducing agent. Parham, P., On the Fragmentation of Monoclonal IgG1, IgG2a, and IgG2b from BALB/c Mice, J Immunol.
- F c has been found to be responsible for much of the non-specific binding of antibody molecules. It is also believed that the molecular weight of the fragments is below the threshold for glomerular filtration, thus allowing for rapid elimination of the fragments. Therefore, one approach to increasing clearance time of antibodies for use in radioimaging has been to break down intact antibody into various fragments, such as Fab and its divalent form, F(ab') 2 . As expected, these fragments are cleared from the body so rapidly that their utility is reduced. Moreover, these fragments may result in reduced uptake by tumor or other target tissue relative to intact antibody.
- Heterobifunctional reagents are reagents having two groups capable of participating in different reactions.
- succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) is heterobifunctional in that its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols.
- the conjugated MAb's used by Orlandi et al. had on average, 11 PDP groups per molecule. Orlandi et al. found that the modified MAb's increase their binding activity in vitro to an extent that molecules not detected by the unmodified MAb's can be detected. These researchers reported no studies of the use of the conjugated MAb's in vivo. Additionally, these researchers believed that molecules having a very low number of antigenic sites were detected by the conjugated MAb's. Accordingly, the PDP modified MAb's had greatly reduced target-cell specificity relative to the unmodified counterparts.
- FIG. 1 shows a schematic representation of the changes believed to occur in a method of producing F(ab') and F(ab') 2 fragments.
- FIG. 2 shows whole body retention of different preparations of radio-labeled MAb's Lym-1 in athymic nude mice.
- FIG. 3 shows the biodistribution as % of injected dose/gram of MAb's Lym-1 and modified Lym-1 in human lymphoma-bearing nude mice seven days after injection.
- FIG. 4 shows the biodistribution as tumor/organ ratios of MAb's Lym-1 and Modified Lym-1 in human lymphoma-bearing nude mice seven days after injection.
- FIG. 5 shows the biodistribution as % of injected dose/gram of MAb's Lym-1 F(ab') 2 and Modified Lym-1 in human lymphoma-bearing nude mice five days after injection.
- FIG. 6 shows the biodistribution as tumor/organ ratio of MAb's Lym-1 F(ab') 2 and Modified Lym-1 in human lymphoma-bearing nude mice five days after injection.
- FIG. 7 shows the image obtained on day 7 after injection of I-131 labeled intact Lym-1.
- FIG. 8 shows the image obtained on day 7 after injection of I-131 labeled modified Lym-1.
- FIG. 9 shows the image obtained on day 7 after injection of I-131 labeled modified Lym-1.
- FIG. 10 shows the image obtained on day 5 after injection of I-131 labeled modified Lym-1.
- FIG. 11 shows the whole body retention of different preparations of radiolabeled monoclonal antibodies B72.3 in athymic nude mice.
- FIG. 12 shows the biodistribution as % of injected dose/gram of MAb's B72.3 and Modified B72.3 in LS174T colon carcinoma-bearing nude mice four days after injection.
- FIG. 13 shows the biodistribution as tumor/organ ratio of MAb's B72.3 and Modified B72.3 in LS174T colon carcinoma-bearing nude mice four days after injection.
- FIG. 14 shows the image obtained on day 1 after injection of I-131 labeled modified B72.3.
- FIG. 15 shows the image obtained on day 4 after injection of I-131 labeled modified B72.3.
- FIG. 16 shows the whole body retention of different preparations of radiolabeled MAb's TNT-1 in athymic nude mice.
- FIGS. 17A-D show a series of bar graphs.
- FIGS. 17A and 17C represent the percentage of injected intact TNT-1 and biotinylated TNT-1 localizing to tumor and various tissues.
- FIGS. 17B and 17D represent ratios of labeled antibodies localizing to tumor and various organs.
- FIG. 18 shows a line graph representing whole-body clearance of intact TNT-1, modified TNT-1 and F(ab') 2 fragments in Balb/c mice.
- FIG. 19 shows a line graph representing whole-body clearance of intact Lym-1, modified Lym-1 and F(ab') 2 fragments in Balb/c mice.
- One aspect of the present invention relates to an antibody conjugated to a chemical reagent at at least one of a plurality of free amino groups disposed on the antibody to produce a modified antibody.
- the antibody has a reduced net positive charge compared to intact antibody.
- the antibody also has an in vivo clearance rate between the clearance rates of F(ab') 2 fragments and intact antibodies of the same type.
- the chemical reagent is not a heterobifunctional agent.
- the antibodies also include a chemical moiety attached thereto.
- the antibody can be a monoclonal antibody or a polyclonal antibody.
- the chemical reagent can be biotin a methyl chelate, such as N 2 S 2 or N 2 S 4 , another chelator, such as EDTA, DPTA or TETA, or a dye, such as FITC.
- the chemical moiety is often a label, such as a radionuclide.
- the radionuclide can be Technicium or halogen radionuclide, such as 125 I or 131 I.
- the label is detectable by magnetic resonance imaging.
- the chemical moiety can be a biologically active molecule, such as a toxin, a drug and a chelate.
- Appropriate drugs include methotrexate, 5-fluoro-uracil, cis-platinum and adriamycin.
- An appropriate toxin is ricin A-chain.
- compositions for immunoscintography includes labeled antibody conjugated with a chemical reagent at free amino groups disposed on the labeled antibody, so that the antibody has a reduced net positive charge compared to intact antibody, and a pharmaceutically excipient, carrier or base acceptable for immunoscintography.
- Yet another aspect of the present invention is a method of preparing a labeled modified antibody having increased antigen binding specificity, decreased non-specific binding and decreased in vivo clearance time.
- the method includes the following steps: obtaining an intact antibody having binding specificity for an antigen to be detected, the native antibody having a plurality of free amino groups disposed thereon, reacting at least one of the free amino groups with a chemical agent to produce a modified antibody, such that the modified antibody has an isoelectric point lower than the isoelectric point of intact antibody, and labeling the modified antibody with a detectable label.
- the method produces a labeled modified antibody.
- the label in this method can be detectable by immunoscintography, such as by a gamma camera.
- Still another aspect of the invention is a method of localizing an antigen in a mammal.
- This method includes obtaining a labeled modified antibody having binding specificity for the antigen to be localized.
- the labeled modified antibody has fewer free amino groups and a reduced isoelectric point compared to an unmodified antibody of the same type, and has incorporated therein a detectable label.
- the labeled modified antibody is administered to the mammal.
- the method allows the antigen and the labeled modified antibody to bind in vivo.
- the labeled modified antibody bound to the antigen is detected, thereby localizing the antigen.
- the antibody can be an intact antibody chemically modified at free amino groups. Such an intact antibody can be chemically conjugated with a heterobifunctional reagent or chemically conjugated with biotin.
- a further aspect of the invention relates to a method of treating a disease state in a mammal.
- This method includes obtaining an intact antibody specific to the diseased tissues in the mammal.
- the intact antibody has disposed thereon a plurality of free amino groups.
- the method also includes modifying at least one of the free amino groups by conjugation with a chemical reagent other than a heterobifunctional reagent to produce a modified antibody.
- the modified antibody has a reduced isoelectric point compared to the intact antibody.
- a biologically active molecule is attached to a first attachment site disposed on the modified antibody other than the site of the chemical reagent. The antibody is then administered to the mammal, thereby treating the disease state.
- methyl chelates such as N 2 S 2 and N 2 S 4
- other chelators such as EDTA, DPTA and TETA
- a number of dyes such as FITC
- Heterobifunctional reagents other than SPDP including sulfosuccinimidyl 2-(p-azido salicylamido)ethyl-1,3'-dithiopropionate (SASD), sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (SAND), sulfosuccinimidyl (4-azidophenyl-dithio)propionate (sulfo-SADP) and 2-aminothiolane•HCl (Traut's reagent), are believed to provide similar results when conjugated with antibodies in accordance with the present invention.
- SPDP sulfosuccinimidyl 2-(p-azido salicylamido)ethyl-1,3'-dithiopropionate
- SAND sulfosuccinimidyl 2-(m-
- modified antibodies of the present invention can be advantageously linked to another chemical moiety to provide a specific diagnostic or therapeutic benefit.
- any of a variety of well known labels such as a radionuclide or an enzyme, can be attached.
- a therapeutic moiety such as an antineoplastic compound or toxin, can also be attached.
- heterobifunctional agents and biotin have been previously used as linkers to attach labels and other moieties to antibodies.
- Biotin itself can function as a label in certain circumstances.
- neither biotin nor heterobifunctional agents have been used with the goal of modifying antibodies to achieve enhanced binding specificity, decreased non-specific binding and decreased in vivo clearance times.
- previous antibodies have not had a modifying agent, such as a heterobifunctional agent or biotin, attached at a first site thereon and an attached label or other chemical moiety at a second site thereon.
- the second attachment site will generally not have an attached modifying agent of the same type as the modifying agent attached at the first attachment site.
- modified antibodies useful in the practice of the invention are chemically modified at free amino groups and are additionally labeled with a detectable label.
- labeling of the modified antibody at a site other than at free amino groups can provide an antibody useful in the practice of the invention.
- a label is chemically conjugated to the antibody, either before or after chemical modification of the antibody at free amino groups, that label can be attached to the antibody at a site other than at a free amino group and at a site other than the site of the amino group-modifying reagent when that reagent is a heterobifunctional reagent.
- tyrosine residues present in the antibody protein can be modified by radioiodination.
- detectable labels that can be attached to antibody tyrosine residues are not limited to iodine.
- Other labels that can be attached to antibody tyrosine residues include halogen radionuclides, such as isotopes of F, Cl, Br, I and others. The attachment of such halogen radionuclides to antibodies is described in Wilbur, Bioconj. Chem., 3:433-470 (1992), the disclosure of which is hereby incorporated by reference. Technicium radionuclides bind to other residues on the antiobody molecule.
- any antibody having a plurality of aminoside moieties modified such that the pI of the of the antibody is reduced relative to the unmodified antibody will exhibit improved target specificity by virtue of having reduced non-specific interactions with non-cognate antigens.
- a second feature of antibodies modified in accordance with the invention renders them particularly useful for in vivo antigen localization.
- tumor/organ ratio Two factors that can improve the signal-to-noise ratio, which can be represented as the "tumor/organ ratio," in antibody-based antigen imaging procedures are: (1) increased tumor localization, and (2) decreased levels of non-specifically bound labeled antibody.
- MAb's chemically modified to have isoelectric points reduced relative to the unmodified intact MAb can advantageously increase binding specificity while reducing non-specific binding and decreasing the whole-body clearance time relative to unmodified antibody.
- a detectable label can, for example, be a radionuclide. More specifically, we have now discovered that antibodies modified to contain biotin moieties exhibit substantially improved capacity for binding antigen. As disclosed below, labeled biotinylated antibodies have been used in a method for localizing tumor cells in vivo. In the practice of the invented method, it is essential for the chemically modified antibody te be labeled directly. This contrasts with methods employing antibodies labeled indirectly as described by Khawli et al. in Antibody, Immunoconjugates, and Radiopharmaceuticals, 6:13 (1993), the disclosure of which is hereby incorporated by reference.
- reagents useful in procedures for improved tumor localization can be produced by obtaining an antibody having binding specificity for a desired target antigen, chemically modifying free amino groups on the antibody using a reagent such as a heterobifunctional reagent or biotin and then labeling the antibody with a detectable label such as a radionuclide.
- a reagent such as a heterobifunctional reagent or biotin
- a detectable label such as a radionuclide.
- the order of the chemical modifying and radiolabeling steps is optional.
- a step for radiolabeling substantially purified antibodies can be eliminated if the antibodies employed in the procedure are MAb's and if the hybridoma producing those MAb's is propagated in growth medium containing labeled precursors that are incorporated into the MAb products of the hybridoma.
- radiolabeled and biotinylated MAb's useful in connection with the invention can be produced by propagating the MAb-producing cell line in growth medium containing radiolabeled amino acids, collecting the radiolabeled MAb's, and biotinylating the radiolabeled MAb's.
- Alternative methods for labeling antibodies, either before or after a biotinylation step, will be apparent to those having ordinary skill in the art.
- biotinylated antibodies exhibit improved targeting when compared with nonbiotinylated antibodies. Since an antibody useful in connection with the invention can be detected by virtue of a label carried on the antibody, the presence of biotin groups on the labeled antibody provides obvious advantages with respect to targeting and detection. Thus, essential features of antibodies useful in connection with the invention and that the antibodies: (1) have aminoside moieties chemically modified such that the pI of the modified antibody is reduced relative to the unmodified antibody, and (2) harbor a label that can be detected by a detection means.
- modified antibodies of the present invention advantageously, have surprisingly enhanced diagnostic and therapeutic effectiveness relative to fragments of antibodies, such as F(ab') or F(ab') 2 .
- the following example shows an exemplary method for the introduction of, on average, one PDP group to a monoclonal antibody.
- Lym-1 with SPDP Lym-1 (IgG 2a )
- the monoclonal antibody against B cell lymphoma was obtained as in Epstein, A. L. et al., Two New Monoclonal antibodies, Lym-1 and Lym-2, Reactive with Human B-lymphocytes and Derived Tumors, with Immunodiagnostic and Immunoreactive Potential, Cancer Res. 47: 830-840 (1987), the disclosure of which is hereby incorporated by reference.
- the Lym-1 MAb's were functionalized using SPDP, a heterobifunctional reagent which reacts with free amino groups on antibodies as in Carlson, J.
- the degree of functionalization of Lym-1 with SPDP was determined to be an average of one PDP group per molecule by measurement of release of pyridine-2-thione at 343 nm after reduction of an aliquot of the Lym-1 solution with molar excess of 7 mg dithioerythritol in phosphate buffer saline solution (PBS), pH 7.2., as in Grassetti, D. R. and Murray, J. F., Determination of Sulfhydryl Groups with 2,2'- or 4,4'-dithiodipyridine, Arch. Biochem. Biophys. 119: 41-49 (1967), the disclosure of which is hereby incorporated by reference.
- Example 2 The modified antibody from Example 1 was analyzed by Fast Protein Liquid Chromatography (FPLC) in order to show that the antibodies remained substantially intact. This analysis is shown in Example 2.
- FPLC Fast Protein Liquid Chromatography
- modified antibody from Example 1 was achieved by Fast protein Liquid Chromatography (FPLC) equipped with a fixed wavelength UV spectrophotometer set at 280 nm. Size exclusion chromatography was performed on a superose-12 column (Pharmacia) with PBS pH 7.2 as the solvent system, eluting at flow rate of 1 mL/min. The modified Lym-1 appeared at a retention time of 690 seconds, identical to the retention time of unlabeled intact Lym-1.
- FPLC Fast protein Liquid Chromatography
- Example 2 shows that the SPDP-modified antibodies behaved virtually identically to the unmodified antibodies in FPLC. This data shows that the modification likely did not lead to breakdown of the intact molecules in vitro.
- radiolabelling of the modified MAb's was performed.
- the radiolabelling is shown in Example 3.
- the reaction was quenched after 3 minutes with 20 ⁇ L of 120 mM solution of sodium metabisulfite.
- the radiolabeled MAb's from Example 3 were subjected to Instant Thin Layer Chromatography (ITLC) in order to determine the purity of the labeled MAb's. This analysis is shown in Example 4.
- ITLC Instant Thin Layer Chromatography
- Modified Lym-1 radiolabeled with 131 I and modified Lym-1 radiolabeled with 125 I via the chloramine T method of Example 3 were analyzed using an analytical ITLC system consisting of silica gel impregnated glass fiber. Strips (2 ⁇ 20 cm) were activated by heating at 110-1/2° C. for 15 minutes prior to use; spotted with 1 ⁇ L of sample; air-dried and eluted with MeOH/H 2 O (80:20) for approximately 12 cm; again air-dried, cut in half and counted to determine protein-bound and non-protein-bound radioactivity. Both forms of radiolabeled Lym-1 antibodies had an R f value of 0 and showed radiochemical purity of ⁇ 99%. Analysis of intact Lym-1 labeled in the same way as in Example 3 revealed the same purity.
- Example 4 shows that high purity radiolabeled antibodies could be obtained.
- the immunoreactivities of these radiolabeled MAb's were tested by their ability to bind to Raji cells. This analysis is shown in Example 5.
- the in vitro immunoreactivities of the radiolabeled modified Lym-1 and intact Lym-1 were evaluated by conventional live assay of 10 6 Raji cells/tube by the method of Epstein, A. L. et al., supra. Briefly, Raji cells resuspended in 100 ⁇ L of 1% bovine serum albumin in PBS was pipetted into a triplicate set of test tubes. One hundred ⁇ L of the labeled Lym-1 was added to each test tube (100,100 cpm/tube) and incubated for 30 minutes at room temperature with continuous mixing using an orbital shaker.
- the cells were washed three times with 1% bovine serum in PBS by spinning the tubes at 1000 rpm for 5 minutes, decanting the supernatant and resuspending the cells in 200 ⁇ L PBS.
- bound Lym-1 was detected by measuring the radioactivity bound to the cells using a gamma counter. The results showed that the binding activity of the modified Lym-1 was 87%, whereas the intact Lym-1, which served as a standard control, had a binding activity of 80%.
- Example 5 shows that the modified Lym-1 was more immunoreactive in vitro than the unmodified Lym-1.
- modified MAb's were analyzed for their stability in serum, as shown in Example 6.
- Monoclonal antibodies of modified Lym-1 and intact Lym-1 which was labeled directly with I-125 were added to each of several triplicate sets of fresh mouse serum to a final concentration of 100 ⁇ g/mL.
- the tubes were incubated at 37-1/2° C. in a humidified incubator maintained in 5% CO 2 in air.
- protein-bound activity was determined by adding 900 ⁇ L of 100% trichloroacetic acid (TCA) to 100 ⁇ L aliquots. After a five-minute incubation at room temperature, protein precipitates were sedimented by centrifugation, and 500 ⁇ L of supernatant were withdrawn from each tube and counted for radioactivity in a gamma counter.
- TCA trichloroacetic acid
- Example 6 showed that the stability of the activity of the modified antibodies was maintained in serum for at least 8 days.
- HPLC analysis of the modified Lym-1 after incubation was performed, as shown in Example 7.
- HPLC analyses were performed on a Waters system equipped with size exclusion columns (SW 300) with 0.1M neutral phosphate buffer as eluting solvent and a flow rate of 1 mL/min. The eluate was detected with a radioisotope detector.
- the labeled modified Lym-1 product mixture from Example 6 revealed one major peak of a low molecular weight species with an elution time of 750 seconds, plus a small quantity at 690 seconds. The intact Lym-1 gave a single peak with a retention time of 690 seconds.
- Example 7 shows that the serum incubated modified Lym-1 samples had an apparent molecular weight in HPLC analysis lower than that of intact Lym-1.
- Example 2 showed that unincubated modified Lym-1 had an identical retention time to intact Lym-1.
- the modified Lym-1 showed an apparent loss of molecular weight in FPLC analysis upon incubation in serum.
- Example 6 The same samples from Example 6 were also examined over an 8-day study to see if there had been any loss of radioactivity from the radiolabeled Lym-1; such loss can be interpreted as evidence of deiodination in serum. The data showed virtually no loss of radioactivity over this period, confirming that a very stable attachment of iodine had been obtained in these immunoconjugates.
- Examples 7-9 show that the modified antibodies while retaining virtually full activity after incubation in serum, appeared to break down into molecules of apparent molecular weight of 116,000. As stated above, it is possible that this loss of molecular weight is due to the breakdown of the antibodies into their monovalent form. In any event, it is believed that the loss in apparent molecular weight is due to the breakdown of the modified antibodies into fragments thereof.
- Example 10 After discovering the foregoing unexpected change in apparent molecular weight of the modified antibodies when incubated in serum, we tested the stability of the modified MAb's in vivo. We performed these in vivo tests in order to determine total body clearance time. An example of these tests is shown in Example 10.
- the clearance of F(ab') 2 fragments was, however, two times faster, with a biological half-life of 10 hours, than the modified Lym-1.
- the data showed that modified Lym-1 is cleared at a rate intermediate between the rapidly-cleared F(ab') 2 fragments and the slowly-cleared intact antibody.
- Example 11 shows the methods used in all of the subsequent biodistribution studies.
- mice received a 0.2 mL inoculum containing 10 ⁇ g of modified Lym-1 labeled with I-131 at 12 ⁇ Ci/ ⁇ g (120 ⁇ Ci/mouse), and 10 ⁇ g of F(ab') 2 fragments labeled with I-125 at 2.5 ⁇ Ci/tig (25 ⁇ Ci/mouse).
- mice were sacrificed by cervical dislocation at preselected times, post-injection, and various organs, blood and tumor were removed and weighed on an analytical balance. The samples were then counted in a gamma counter to determine the 131 I and 125 I activity.
- 125 I counts were adjusted for cross-over from the 131 I channel by subtracting 17% of 133 I channel counts, a formula that was determined experimentally using a 1282 Compugamma gamma counter (LKB). The data were also corrected for the radiation decay of the 131 I isotope according to the days on which the animals were sacrificed. For each mouse, data was expressed as cpm per gram tumor/cpm per gram organ, % dose/gram, and % dose/organ From these data, the mean and standard deviation were calculated for each group.
- LLB Compugamma gamma counter
- Example 12 compares the biodistribution of the modified MAb's to intact MAb's using the methods of Example 11.
- Example 11 the intact Lym-1 antibody was compared to the modified Lym-1 antibody in the methods of Example 11. Intact Lym-1 produced a blood activity of 0.64% ID/g at 7 days after injection, as reported in Table I. At the end of the same time interval, the tumor had an activity of 3.92% ID/g.
- the modified Lym-1 cleared from blood faster and produced a blood activity of 0.14% ID/g at 7 days. At the end of the same time interval, the tumor produced 7.7%, which tended to be significantly higher than the corresponding activities of the intact Lym-1.
- the modified antibodies produced a higher signal in the tumor than the intact antibodies. Additionally, the modified antibodies reacted less strongly than the intact MAb's for every organ tested, except for the kidney. It is not unexpected that a higher signal would be found in the kidney, because the antibodies are expected to be cleared through this organ. Due to the more rapid clearance rate of the modified MAb's relative to intact MAb's found in Example 10, a higher amount of modified MAb's in the kidney would be expected.
- the modified MAb's produced a significantly higher tumor/organ ratio than intact MAb's in every organ tested, except for kidney.
- the modified antibodies would produce a significantly lower background when used in immunoscintography.
- the modified antibodies would be more effective when used in immunotherapies due both to its higher affinity for tumor and lower affinity for non-target tissues.
- the modified antibodies would, thus, be expected to be more highly toxic to tumors and less toxic to non-target tissues.
- Example 13 is illustrative of these experiments.
- Table II shows that the modified Lym-1 cleared more slowly from the blood than the F(ab') 2 fragments.
- the modified Lym-1 produced a blood activity of 0.09% ID/g higher than the fragments (0.05%) at 5 days post-injection.
- FIG. 5 shows that the tumor activity of the modified Lym-1 was about two-and-one-half times higher than the corresponding activity of the F(ab') 2 fragments.
- the activity of the modified Lym-1 was higher than the F(ab') 2 fragments for all of the various organs tested, including kidney. This is consistent with the theory that more rapidly cleared antibodies accumulate in the kidney.
- FIG. 6 shows that the tumor-to-organ ratios for modified Lym-1 are higher than those of the F(ab') 2 fragments for all of the organs tested.
- the experiments of Examples 12 and 13 confirm that the modified antibodies of the present invention have a higher activity for their target tumor than either intact MAb's or F(ab') 2 fragments. Additionally, the tumor-to-organ data of these experiments shows that the modified antibodies have higher specificity for tumor than either the intact MAb's or F(ab') 2 fragments.
- Tumor-bearing nude mice were imaged using a pinhole collimator and a spectrum 91 gamma camera (Raytheon). Image analyses of these animals provided an estimate of tumor/whole body antibody distribution after injection. Seven days after injection, the mice were anesthetized with 2 mg Ketamine HCl and 0.4 mg Xylazine administered as a 0.2 mL s.c. inoculation. The immobilized mice where then imaged in a posterior position with the camera preset to record 10,000 counts. No background subtraction was performed. Photographic images were obtained using Polaroid Type 330 Pack film. Two areas in each image were defined: (a) region 1, whole body; (b) region 2, tumor. FIGS. 6-8 show exemplary scintographs (also known as scintograms) produced by these experiments.
- FIG. 6 shows that although the tumor was visualized, the rest of the animal was also visualized.
- FIGS. 7 and 8 show the images of two different Raji tumor-bearing animals injected with labeled modified Lym-1 at the same time after injection. It can be seen that both FIGS. 7 and 8 show concentration of the labeled modified Lym-1 at the tumor at levels much higher than thliose at the tumor produced by the intact Lym-1, seen in FIG. 6. More importantly, the ratio of label at the tumor to the background of the whole mouse produced by the modified Lym-1 was several times higher than that of the intact Lym-1. Thus, FIGS. 7 and 8 show a clear definition of the tumor, with little or no background radioactivity.
- FIG. 10 shows a 5-day image taken of the same animal as shown in FIG. 9 at 7 days. As can be seen, the 5-day image of FIG. 10 was significantly superior to the image produced by intact Lym-1 at 7 days (FIG. 7). Results were similar for all animals tested.
- modified antibody fragments exhibit greater specific activity to tumor antigens, allowing more absolute concentration of antibody to accumulate in tumor. This is confirmed by our results that showed that the absolute concentration of modified Lym-1 fragments is about 2 times the intact Lym-1 concentration 7 days after injection and about two and a half times the F(ab') 2 fragments at five days.
- the much faster clearance of the modified Lym-1 fragments also significantly decreases the time required to reach high tumor to background ratios and thus results in better imaging in less time than intact antibody.
- B72.3 (IgG 1 ), the monoclonal antibody against colon carcinoma, was obtained as in Coicher, D. et al, A Spectrum of Monoclonal antibodies Reactive with Human Mammary Tumor Cells, Proc. Natl. Acad. Sci. 78:3199-3203 (1981), the disclosure of which is hereby incorporated by reference.
- B72.3 MAb's were functionalized with an average of one PDP group per molecule according to the method of Example 1.
- the modified B72.3 MAb's were radiolabeled by the method of Example 3. Total body clearance times were measured as in Example 10.
- FIG. 11 shows the results of these total body clearance experiments.
- the modified antibodies showed a decrease in whole body half-time clearance from the approximately 6 days of intact MAb's to approximately 2.5 days for the modified antibodies.
- the half-time clearance of F(ab') 2 fragments was, as for the Lym-1 fragments, faster than the modified antibodies, with a half-time of approximately 12 hours.
- the results showed that modified B72.3 behaved similarly to the modified Lym-1 in having a half-time clearance intermediate between that of the F(ab') 2 fragments and intact antibody.
- intact B72.3 antibody produced a blood activity at 1.34% ID/g at 4 days after injection, and an activity of 4.04% at the tumor, as shown in Table III.
- the modified B72.3 produced lower blood activity (1.1% ID/g) and higher tumor activity ((6.02% ID/g) at 4 days.
- the tumor to organ ratio for modified B72.3 was significantly higher than the corresponding ratios for the intact B72.3.
- the tumor to organ ratio was even improved for kidney due to the higher activity of the modified antibody at the tumor site.
- FIG. 14 shows an immunoscintograph at 1 day after injection. The image shows a clear definition of the tumor with little background radioactivity.
- FIG. 15 shows an immunoscintograph at 4 days after injection. By 4 days, the tumor was clearly seen with little radioactivity remaining in the blood pool of the animal. Results were similar for all animals.
- the modified B72.3 was found to be very useful in obtaining high quality immunoscintographs within a short time of injection of tumors reactive with B72.3.
- TNT-1 is an IgG 2a monoclonal antibody which utilize necrotic tumor as a target for their selective binding to human cancers. We modified this antibody with on average one PDP group per molecule as in Example 1, and analyzed whole body retention time as shown in Example 18.
- TNT-1 as in Epstein, A. L. et al., A Novel Method for the Detection of Necrotic Lesions in Human Cancer, Cancer Res. 48:5842-5848 (1988), the disclosure of which is hereby incorporated by reference.
- the TNT-1 MAb's were radiolabeled by the method of Example 3. Total body clearance times were measured as in Example 10.
- FIG. 16 shows the results of these total body clearance experiments.
- the modified TNT-1 MAb's showed a decrease in whole body half-time clearance time relative to intact TNT-1, and an increase relative to the F(ab') 2 fragments of TNT-1.
- the modified TNT-1 behaved similarly to the other modified antibodies. We, therefore, expect, that the utility of the modified TNT-1 MAb's to be equivalent to the other modified antibodies tested.
- Example 19 describes one method useful for preparing biotinylated antibodies.
- the TNT-1 and Lym-1 MAb's were separately conjugated to 6-(biotinamido) hexanoate by reaction with its sulfo N-hydroxysuccinimide ester (NHS-LC-biotin).
- NHS-LC-biotin sulfo N-hydroxysuccinimide ester
- a standard solution of 2 mg NHS-LC-biotin in 1 mL of 0.9% saline solution was prepared.
- the reagent mixture was incubated for 2.5 hours at room temperature with continuous stirring at low speed.
- the coupled antibody was chromatographed on a PD-10 column (Pharmacia) equilibrated with PBS, pH 7.2. Purity of the coupled antibody preparations was assessed by FPLC using a superose-12 column. The results of these procedures indicated that the biotinylated antibody was obtained with at least 99% purity.
- the average number of biotin groups coupled to each antibody molecule was determined spectrophotometrically according to the method described by Green in Biochem J. 94:23c-24c (1965). Briefly, the biotinylated antibody was digested enzymatically with 1% protease at 37° C. for four hours. To a 5 mL solution containing 800 ⁇ L of 100 ⁇ M HABA in 0.1 M PBS, pH 7.2 was added 70 ⁇ L of a 17 ⁇ M solution of streptavidin. The streptavidin-HABA solution was then titrated with increasing volumes of the digested biotinylated antibody solution, and the change in absorbance determined at 500 nm. From this treatment, the concentration of biotin in the protease-treated antibody solution was calculated using a standard curve of biotin solution. The results indicated that an average of from 3 to 4 biotin moieties were incorporated into each antibody molecule.
- biotin-antibody conjugates were radiolabeled with 125 I using the chloramine-T method essentially as described in Example 3.
- Example 20 describes the methods used for radioiodination of antibodies and antibody fragments.
- ITLC analytical instant thin layer chromatography
- radiolabeled biotinylated antibodies prepared according to the procedure described above were stored at 4° C. and administered to mice within four hours of labeling.
- Example 21 describes the methods used to demonstrate that antibodies having been modified by biotinylation and radiolabeling retained both antigen-binding ability and structural integrity.
- Immunoreactivity of the modified TNT-1 MAb was evaluated using a fixed cell radioimmunoassay described by Gaffar et al. in J. Immunoassay, 2:11 (1991). Briefly, radiolabeled TNT-1 and radiolabeled biotinylated TNT-1 were incubated for 30 minutes with Raji cells previously fixed with 20% paraformaldehyde in PBS at room temperature and then treated with acetone at -20° C. The cells were then washed with 1% BSA in PBS and counted in a gamma counter. The results of this procedure indicated that approximately 60% of both antibody preparations bound to the fixed cells. This indicated that the antigen binding activity of radiolabeled biotinylated TNT-1 was comparable to that of the radiolabeled intact TNT-1 which served as a standard control.
- Example 22 describes the methods used to prove that radiolabeled biotinylated antibodies were not unusually subject to degradation in the presence of serum.
- MAb's labeled directly with 125 I were added to a triplicate set of tubes containing fresh mouse serum to a final concentration of 100 ⁇ g/mL. All samples were incubated at 37° C. in a humidified incubator maintained at 5% CO 2 in air. At various times between 0 and 8 days, protein-bound radiolabel was determined by adding 900 ⁇ L of 100% trichloracetic acid (TCA) to 100 ⁇ L aliquots of each sample, incubating at room temperature for five minutes and recovering protein precipitates by centrifugation. Aliquots (500 ⁇ l) of supernatant were withdrawn from each tube and counted for radioactivity using a gamma counter.
- TCA trichloracetic acid
- radiolabeled biotinylated antibody was stable in vitro at all time points. More specifically, at least 97% of the radiolabel remained protein-associated after 8 days of incubation. This confirmed that the biotin moieties present on the MAb's had no detrimental effect on the stability of the protein-associated radiolabel.
- Isoelectric focusing in polyacrylamide gels was performed in a BioRad Model 111 Mini IEF cell.
- Samples were electrophoresed through a pH gradient constructed with a mixture of BioLyte ampholytes (BioRad) at concentrations of 1.2% 3/10 ampholyte and 0.8% 5/8 ampholyte according to protocols provided by BioRad.
- IEF standards BioRad
- IEF gels were stained with Coomassie Blue R-250 and dried overnight. The results of these procedures confirmed that biotinylation of the MAb's substantially altered the electrical charge properties of the macromolecules.
- the radiolabeled Lym-1 had a pI value of 7-8
- the radiolabeled biotinylated Lym-1 had a pI of 5-6
- the radiolabeled TNT-1 MAb had a pI value of 5.5-6.5
- the radiolabeled biotinylated TNT-1 MAb had a pI of 4.5-5.0.
- Example 23 describes the methods used to demonstrate that MAb's having aminoside moieties chemically modified to result in a macromolecule having reduced pI relative to native antibody advantageously exhibited: (1) increased target specificity, (2) decreased non-specific binding and (3) decreased clearance time.
- mice Paired-label studies.
- each mouse was injected intravenously with a 0.2 mL inoculum containing 120 ⁇ Ci/10 ⁇ g of modified 131 I-labeled MAb and 25 ⁇ Ci/10 ⁇ g of intact I 125 I-labeled MAb.
- mice received a 0.2 mL inoculum containing 120 ⁇ Ci/10 ⁇ g of modified 131 I-labeled MAb and 25 ⁇ Ci/10 ⁇ g of 125 I-labeled MAb F(ab') 2 .
- mice were sacrificed by cervical dislocation at preselected times post-injection and various organs, blood, and tumor were removed and weighed. The samples were then counted in a gamma counter to quantitate the 131 I and 125 I activities. 125 I counts were adjusted for crossover from the 131 I channel by subtracting 17% of the 131 I channel counts, a formula that was determined experimentally for the 1282 CompuGamma counter. The data were also corrected for the radiation decay of the 131 I isotope according to the time when the animals were sacrificed. For each mouse, data are expressed as cpm per gram tumor/cpm per gram organ and % dose/gram. From these data, the mean and standard deviation were calculated for each group. The same paired-label biodistribution studies were performed using Lym-1 in the Raji lymphoma model and B72.3 in the LS-174T human colon carcinoma model.
- the advantage of the modified antibody is represented quantitatively in FIG. 17B, wherein the ratio of tumor-localized antibody to non-specifically localized antibody is presented for each tissue.
- the advantage of the biotinylated antibody as an imaging reagent was particularly evident in muscle and pancreas.
- FIG. 17C shows that biotinylated TNT-1 antibody localized to tumor tissue more efficiently than the non-biotinylated counterpart antibody. At the same time, the amount of non-specific binding was advantageously lower for the biotinylated species.
- FIG. 17D quantitatively confirms that the tumor/organ ratio, which reflects the signal-to-noise ratio in various tissues, was highest in muscle and pancreas. Notably, FIGS. 17C and 17D include results obtained using TNT-1 F(ab') 2 fragments which have rapid whole-body clearance rates.
- FIGS. 18 and 19 disclose the results of whole-body clearance rate studies for TNT-1 and Lym-1 antibodies and their derivatives.
- the results presented in FIG. 18 indicated that TNT-1 MAb modified by either SPDP or biotin have decreased clearance times relative to the intact antibody.
- the F(ab') 2 TNT-1 fragment, which had the fastest clearance rate of all antibodies tested was used as a positive control in this procedure.
- the biotinylated TNT-1 MAb advantageously exhibited a more rapid clearance rate than the non-biotinylated antibody.
- the results presented in FIG. 19 indicated that Lym-1 MAb modified by either SPDP or biotin had decreased clearance times relative to the intact antibody. This illustrated that agents that chemically modified free amino groups moieties to reduce the pI of the antibody advantageously increased the antibody binding specificity and whole-body clearance rate.
- any antibody using the methods of the present invention will provide a reagent useful for improved tumor imaging.
- antibodies to that tissue type must first be obtained.
- Polyclonal antibodies can be obtained in a conventional manner as will be known by one of skill in the art.
- monoclonal antibodies can be prepared in order to obtain the increased specificity provided by these antibodies, as will also be known by one of skill in the art.
- the antibodies are then chemically modified at free amino groups by conjugation with a heterobifunctional agent, biotin, or other agent that will lead to a modified antibody product having an isoelectric point that is lower than the isoelectric point of the unmodified antibody. After conjugation, a suitable label is applied to the modified antibodies.
- radio-opaque materials such as barium, cesium or iodine can be imaged using conventional X-rays.
- Paramagnetic or supermagnetic particles can be used as labels, using MRI imaging technology to produce an image of the location of the antibodies.
- technicium can be used as label.
- the labeled antibodies can be included in pharmaceutical preparations for the introduction of label into a subject including pharmaceutically acceptable excipients, carriers, or bases.
- suitable excipients, carriers, or bases include saline, phosphate buffered saline, glycerol, calcium carbonate, and the like.
- These compositions are then introduced through any of a variety of means, such as local injection, intravenous injection, or oral administration in cases where reduced signal strength is required or where imaging of tissue in the oral cavity are desired. However, preferably, administration is through systemic injection in order to maximize exposure of the targeted tissue to the antibody.
- Such therapeutic agents generally comprise an antibody specific to a tumor or other diseased tissue combined with one or more biologically active molecules.
- Suitable biologically active molecules which function in such agents are toxins, such as the diphtheria toxin (ricin) A-chain or any of a variety of plant toxins known by those of skill in the art; radionuclides, such as radioactive isotopes of yttrium, iodine, phosphorus, and other commonly used radio-therapeutic agents; drugs, such as methotrexate, 5-fluoro-uracil, or adriamycin; chelates, including EDTA and EGTA; cis-platinum and other toxic organo-metallic agents, and any other therapeutic agent.
- toxins such as the diphtheria toxin (ricin) A-chain or any of a variety of plant toxins known by those of skill in the art
- radionuclides such as radioactive isotopes of yttrium, iodine, phosphorus, and other commonly used radio-therapeutic agents
- drugs such as methotrexate, 5-fluoro-
- antibodies specific to particular undesired tissue types must first be obtained. If the desired antibodies are not available, the antibodies may be raised in a suitable organism by injecting the organism with antigens and obtaining serum from the mammal, as will be known by one of skill in the art. Alternatively, and preferably, monoclonal antibodies can be raised in a manner known to one of skill in the art. The antibodies are then chemically conjugated with an agent such as a heterobifunctional agent, biotin or other agent capable of modifying free amino groups present on the antibody molecule. After conjugation, the resulting modified antibodies are further modified by conjugation with a biologically active agent, such as a therapeutic agent or detectable label as described above.
- a biologically active agent such as a therapeutic agent or detectable label as described above.
- compositions containing a pharmaceutically acceptable carrier, excipient or base.
- pharmaceutically acceptable carriers, excipients, or bases include normal saline for systemic injection, glycerol, calcium carbonate.
- the compositions are then ready for introduction into a patient, such as a mammal.
- compositions can be introduced through systemic injection, local injection into the affected tissue, can be applied topically to externally affected tissue, and can be taken orally in cases where reduced signal strength is required or where therapy of tissue in the oral cavity are desired.
- Dosage of the biologically active agent containing antibody will depend on target tissue sensitivity to the toxin, the amount of affected tissue, route of administration, the affinity of the antibody, clearance rates and on other factors. However, representative dosages will generally be in the range from 1 ⁇ g/kg total body mass to 1 mg/kg. In most applications, the dose will preferably be from 5 to 200 ⁇ g/kg.
- Example is illustrative of an immunotherapy effective against Raji tumors in mice.
- PDP-modified antibodies are employed in the Example, antibodies having free amino groups modified by other agents, such as biotin, that will produce a modified antibody having a pI lower than a corresponding intact antibody are expected to work equally well.
- PDP-modified Lym-1 is obtained as in Example 1.
- the modified antibody is then treated to introduce, on average, one ricin A-chain per antibody molecule.
- Intact Lym-1 and F(ab') 2 fragments are similarly combined with toxin.
- mice Twenty-five mice are divided into five groups.
- Group I receives intraperitoneal injections at 10 ⁇ g/kg total body weight of the ricin-PDP-modified Lym-1 in phosphate buffered saline (PBS) once per week for 8 weeks.
- Group II receives injections of an equivalent amount ricin-intact Lym-1.
- Group III receives equivalent amounts of ricin-F(ab') 2 fragments of Lym-1.
- Group IV receives an equivalent amount of unconjugated ricin.
- Group V receives PBS alone.
- mice After 8 weeks, immunoscintography of all surviving mice using the method of Example 14 is performed.
- the Group I mice show reduced visualization of tumor compared to any of the other groups.
- Surviving Group II and Group III mice show some improvement, though less dramatic than the Group I mice.
- Group IV mice become very ill or die.
- Example 24 shows one particular treatment of a tumor using the modified antibodies of the present invention.
- Example 19 shows the superior results achieved when using the PDP-modified antibodies of the present invention. Substituting the use of other antibodies specific to other tumors or diseased tissues in mice or other mammals, such as humans, is believed to produce similarly effective results in treating those specific tumors or diseased tissues. Moreover, the substitution of other know toxins is believed to also produce similarly effective results.
- Example 20 shows the use of a similar therapy effective against pancreatic cancer in humans. Although PDP-modified antibodies are employed in the Example, antibodies having free amino groups modified by other agents, such as biotin, that will produce a modified antibody having a pI lower than a corresponding intact antibody are expected to work equally well.
- a monoclonal antibody is obtained which is specific to an antigen found in human pancreatic tumors.
- This antibody is modified by conjugation to, on average, one PDP group per antibody molecule, as in Example 1. Methotrexate is then conjugated to these modified antibodies as described for Ricin in Example 19.
- the first group receives intravenous injections of the drug-PDP-MAb in PBS at 20 ⁇ g/kg total body weight on a weekly basis in combination with traditional therapy.
- the second, group receives injections of PBS in combination with traditional therapy as a control. After 10 weeks, immunoscintography of the surviving patients is performed.
- the average size of the tumors imaged in the first group of patients is reduced relative to the control group.
- the modified antibodies are formulated into pharmaceutical compositions.
- the PDP-modified antibodies which are conjugated with a drug for immunotherapy may be incorporated into an injectable composition having a cytotoxicity effective amount of the modified antibody-toxin conjugates of the present invention.
- the following is an-example of a cytotoxicity effective composition effective against B-cell lymphomas in humans.
- radiolabeled modified MAb's may be formulated into compositions effective to visualize their specific antigens in immunoscintography.
- compositions effective to visualize their specific antigens in immunoscintography The following is one example of such a composition.
- Example 23 illustrated one method for localizing radiolabeled biotinylated antibodies, those having ordinary skill in the art will appreciate that alternative methods for localizing labeled antibodies will also be applicable.
- the distribution of radioiodinated biotinylated antibodies can be localized by immunoscintographic imaging exactly as described in Example 14.
- radioiodinated biotinylated MAb having binding specificity for a tumor antigen would be useful in diagnostic procedures.
- the following Example describes how such an imaging procedure could be carried out.
- Example 28 describes one method wherein radiolabeled biotinylated MAb's can be used to image tumor cells in vivo.
- a human patient having been diagnosed with metastatic melanoma is first identified. Immunohistological analysis indicates that the patient's melanoma expresses a cell surface antigen stainable with an anti-melanoma MAb.
- the anti-melanoma MAb is first chemically modified by biotinylation of free amino groups according to the method of Example 19 and radioiodinated with 131 I according to the method of Example 20. Substantially purified radiolabeled biotinylated anti-melanoma MAb is then combined with a pharmaceutically acceptable excipient and administered to the patient. After three days, the injected modified MAb's have substantially localized to cells expressing melanoma antigens.
- the localized MAb's are then visualized by immunoscintographic imaging using a pinhole collimator and a spectrum 91 gamma camera obtainable from Raytheon.
- the photographic record indicates a small area of radioactivity on the patients skin, thereby identifying a secondary tumor.
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Abstract
Description
TABLE I ______________________________________ BIODISTRIBUTION OF MODIFIED AND INTACT MONOCLONAL ANTIBODY LYM-1 IN RAJI TUMOR-BEARING NUDE MICE (N = 6) 7 DAYS AFTER INJECTION cpm/g tumor Organ cpm/g organ % dose/g % dose/organ ______________________________________ Modified Lym-1 Blood 140.07(81.30)* 0.14(0.20) -- Skin 93.98(43.40) 0.09(0.04) -- Muscle 364.53(232.97) 0.03(0.03) -- Bone 126.96(55.86) 0.06(0.02) -- Heart 137.34(67.96) 0.07(0.04) 0.01(0.00) Lung 28.31(10.34) 0.28(0.10) 0.06(0.02) Liver 96.80(49.03) 0.09(0.05) 0.15(0.08) Spleen 12.02(5.62) 0.79(0.53) 0.03(0.01) Pancreas 286.43(159.92) 0.04(0.02) 0.00(0.00) Stomach 71.21(30.72) 0.11(0.03) 0.02(0.01) Intestine 133.31(80.82) 0.07(0.04) -- Kidney 17.63(7.63) 0.45(0.12) 0.14(0.03) Tumor 7.70(3.95) 2.98(1.71) Intact Lym-1 (Control) Blood 30.72(17.74) 0.64(1.26) -- Skin 8.83(3.05) 0.41(0.20) -- Muscle 44.39(26.16) 0.15(0.21) -- Bone 19.49(6.08) 0.21(0.18) -- Heart 28.79(13.76) 0.19(0.22) 0.02(0.02) Lung 16.98(8.22) 0.36(0.46) 0.07(0.01) Liver 11.84(5.95) 0.37(0.25) 0.59(0.46) Spleen 3.93(3.74) 1.52(1.14) 0.06(0.03) Pancreas 29.35(12.88) 0.16(0.17) 0.02(0.02) Stomach 11.00(4.55) 0.32(0.11) 0.07(0.03) Intestine 18.06(8.79) 0.23(0.13) -- Kidney 22.44(10.61) 0.20(0.17) 0.06(0.05) Tumor -- 3.92(3.11) 1.02(0.27) ______________________________________ *Mean (standard deviation).
TABLE II ______________________________________ BIODISTRIBUTION OF MODIFIED AND INTACT MONOCLONAL ANTIBODY LYM-1 IN RAJI TUMOR-BEARING NUDE MICE (N = 4) 5 DAYS AFTER INJECTION cpm/g tumor Organ cpm/g organ % dose/g % dose/organ ______________________________________ Modified Lym-1 Blood 39.59(14.84)* 0.09(0.03) -- Skin 13.69(3.15) 0.24(0.06) -- Muscle 75.32(16.22) 0.04(0.01) -- Bone 26.79(7.18) 0.12(0.04) -- Heart 44.42(11.34) 0.08(0.04) 0.01(0.01) Lung 15.78(3.88) 0.21(0.07) 0.04(0.01) Liver 12.19(4.25) 0.29(0.12) 0.27(0.10) Spleen 2.68(1.01) 1.34(0.55) 0.07(0.03) Pancreas 42.17(11.23) 0.08(0.03) 0.01(0.00) Stomach 14.16(4.43) 0.24(0.07) 0.05(0.02) Intestine 28.36(9.96) 0.12(0.05) -- Kidney 12.53(3.15) 0.27(0.09) 0.09(0.03) Tumor -- 3.18(0.89) 3.16(1.09) F(ab').sub.2 Fragments (Control) Blood 29.27(13.17) 0.05(0.02) -- Skin 10.54(2.78) 0.12(0.02) -- Muscle 55.50(14.49) 0.02(0.01) -- Bone 23.73(7.89) 0.06(0.02) -- Heart 35.19(11.01) 0.04(0.02) 0.00(0.00) Lung 12.57(3.69) 0.10(0.03) 0.02(0.00) Liver 10.43(4.20) 0.13(0.05) 0.12(0.04) Spleen 2.59(1.03) 0.54(0.21) 0.03(0.01) Pancreas 31.55(9.82) 0.04(0.02) 0.00(0.00) Stomach 8.03(3.31) 0.17(0.06) 0.03(0.01) Intestine 22.27(7.93) 0.06(0.02) -- Kidney 10.05(2.71) 0.13(0.04) 0.04(0.01) Tumor -- 1.23(0.24) 1.42(0.48) ______________________________________ *Mean (standard deviation).
TABLE III ______________________________________ BIODISTRIBUTION OF MODIFIED AND INTACT MONOCLONAL ANTIBODY B72.3 IN THE HUMAN LS174T COLON CARCINOMA-BEARING NUDE MICE (N = 5) 4 DAYS AFTER INJECTION cpm/g tumor Organ cpm/g organ % dose/g % dose/organ ______________________________________ Modified B72.3 Blood 6.16(2.32)* 1.10(0.45) -- Skin 20.81(3.92) 0.31(0.12) -- Muscle 61.58(16.16) 0.11(0.05) -- Bone 65.25(15.04) 0.10(0.04) -- Heart 31.41(18.44) 0.24(0.11) 0.03(0.02) Lung 11.89(3.25) 0.54(0.18) 0.14(0.04) Liver 21.61(10.36) 0.33(0.14) 0.43(0.17) Spleen 37.89(15.00) 0.18(0.09) 0.02(0.01) Pancreas 60.23(23.73) 0.12(0.06) 0.02(0.01) Stomach 37.74(9.20) 0.17(0.05) 0.04(0.01) Intestine 68.31(28.27) 0.10(0.04) -- Kidney 24.56(10.07) 0.29(0.13) 0.09(0.04) Tumor -- 6.02(1.33) 6.45(1.53) Intact B72-3 (Control) Blood 3.43(1.13) 1.34(0.60) -- Skin 10.44(1.80) 0.41(0.15) -- Muscle 31.78(8.86) 0.14(0.05) -- Bone 33.36(9.84) 0.14(0.06) -- Heart 16.57(8.28) 0.30(0.14) 0.04(0.02) Lung 6.42(1.59) 0.68(0.28) 0.18(0.08) Liver 11.85(4.78) 0.39(0.16) 0.52(0.24) Spleen 18.94(5.61) 0.24(0.12) 0.02(0.01) Pancreas 29.80(9.42) 0.15(0.05) 0.02(0.01) Stomach 18.88(3.83) 0.22(0.04) 0.05(0.01) Intestine 35.61(13.48) 0.13(0.06) -- Kidney 15.20(6.40) 0.33(0.18) 0.11(0.06) Tumor -- 4.04(0.84) 4.28(0.78) ______________________________________ *Mean (standard deviation).
______________________________________ 10 mg/mL Modified radiolabeled Lym-1 from Example 18 Balance Phosphate Buffered Saline (0.9%) ______________________________________
______________________________________ 10 mg/mL Modified radiolabeled B72.3 from Example 15 Balance Phosphate Buffered Saline (0.9%). ______________________________________
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